Membrane interaction is a key factor for the direct activity of AMPs. Many AMPs cause disruption of the physical activity of the microbial membrane and/or translocation across the membrane into the cytoplasm of bacteria to act on an intracellular target. Some of them are presented in Table 1. AMPs kill bacteria by disturbing membrane integrity through membrane lysis (i.e., colistin, bacitracin, daptomycin, and polymyxin B), membrane poration (i.e., gramicidin D and tyrothricin), or the inhibition of cell wall synthesis (i.e., teicoplanin and vancomycin) (Table 1). Gram-negative and Gram-positive bacteria have molecules at the surface that confer a negative charge, allowing an electrostatic interaction with cationic peptides. Thus, teichoic acids in the cell wall of Gram-positive bacteria and lipopolysaccharides in the outer membrane of Gram-negative bacteria provide additional electronegative charge to the bacterial surface. Several models have been proposed to explain the interaction of AMPs with the bacterial membrane. Thus, three possible pathways to disrupt the inner membrane have been described: (i) in the barrel-stave model, peptides can perpendicularly insert into the membrane, promoting peptide–peptide interactions thanks to the AMP's amphipathic structure and resulting in the formation of a peptide-lined transmembrane pore, (ii) concerning the toroidal-pore model, the insertion of the peptides is induced by a curvature in the lipid layer, and a pore is generated by both the peptide and the phospholipid head group, and (iii) in the carpet-model, the peptide is absorbed onto the membrane, covering the entire surface leading to the loss of the membrane integrity and the formation of micelles (Figure 1). Moreover, peptides can translocate into the cytoplasm and directly inhibit the cell wall and protein synthesis, bacterial cell division, or DNA replication by interacting with specific proteins involved in the biological process (18, 19, 48, 50).

Some AMPs may present their activities against viruses. Antiviral peptides (AVPs) can cause membrane instability by integrating into viral envelopes. Both enveloped RNA and DNA viruses can be targeted by AVPs. AVPs can (i) interact with different glycoaminoglycans present on the cell surface competing with the virus for cellular binding sites, (ii) block the viral entry into the cell, (iii) suppress the cell fusion by interfering with the activity of ATPase protein, (iv) suppress viral gene expression, or (v) interfere with the assembly process of the viruses (13, 18, 19). As examples among the AVPs approved by the FDA, the polypeptide enfuvirtide that is a membrane fusion inhibitor block virus (HIV-1) from entering the host cells and azatanavir, an inhibitor of HIV-1 proteases, preventing the maturation of the proteins needed to assemble the viral capsid, can be cited (Table 1).

Some AMPs pass through the fungal membrane by pore formation or act on beta-glucan or chitin synthesis and others interact with the membrane and cause cell lysis of fungi. They can lead to fungi death by (i) the inhibition of DNA, RNA, or protein synthesis, (ii) induction of apoptosis, (iii) permeabilization of membrane, and (iv) inhibition of cell wall synthesis and enzyme activity (18). Thus, the cyclic lipopeptides (anidulafungin and caspofungin) that are used as antifungal drugs are the inhibitors of the beta-(1,3)-D-glucan synthase (Table 1).

In addition to a broad spectrum of antimicrobial activities, AMPs have anti-inflammatory and immunomodulatory properties. They are described as the effective modulators of inflammation and neutralizers of toxins. AMPs can indirectly promote pathogen clearance of the host by stimulating chemotaxis (by recruiting/activating immunocytes), immune cell differentiation, and the initiation of adaptive immunity, while also preventing harmful inflammation and sepsis by the inhibition of cytokine release and direct scavenging of bacterial endotoxins (20, 50). Indeed, some AMPs modulate host immunity by influencing Toll-like receptor (TLR) recognition of microbial products (i.e., the neutralization of bacterial products such as lipopolysaccharide and lipoteichoic acid to suppress inflammation) and nucleic acids released upon tissue damage to promote auto-inflammation (51) (Figure 1). AMPs having immunomodulatory activities belong to the two major families: defensins and cathelicidins. Human defensins exhibit chemotactic properties and are produced by multiple cell types (i.e., neutrophils, macrophages, lymphocytes, and intestinal epithelial cells). The human cathelicidin membrane disrupting peptide LL-37 acts as a chemoattractant for monocytes, neutrophils, mast cells, and T cells (11, 49, 51). In addition, the immunomodulatory properties of several marine-derived AMPs have been demonstrated. Thus, defensins from oyster or mytilus are the AMPs acting as host defense peptides that disrupt the membrane of microbial pathogens and play a major role in immunomodulation by acting on the innate and adaptive immune response (29). Among the approved AMPs that are used as antibacterial agents, the glycopeptide vancomycin exhibits pharmacological activity against Gram-positive bacteria and immunomodulatory activity affecting tumor necrosis factor-alpha (TNF-α) pathways. Thus, Arbabanel et al. showed that oral vancomycin can be used as an effective treatment for concomitant primary sclerosing cholangitis and inflammatory bowel disease in pediatric. Indeed, oral vancomycin-mediated disease resolution is associated with elevated peripheral TGF-β levels without alterations in Th1 or Th2 cytokine production patterns and increased regulatory T-cell levels (52).

Some peptides are already used or currently used in clinical trials to treat local diseases of the gastrointestinal tract, including colon infection disease such as amoebiosis and C. difficile or Enterococcus infections. First, some AMPs present an interest after oral administration without pharmaceutical forms. For example, Stiefel et al. (54) administered ramoplanin, a glycolipodepsipeptide, in drinking water (at 100 μg/ml of water) in female mice and demonstrated that it could potentially be used to reduce cross-transmission of vancomycin-resistant Enterococcus. Nevertheless, this work demonstrated some limitations notably due to the administration of this AMP in drinking water that, for example, may not reproduce the pharmacokinetic of human dosing.

LFF571, a new investigational thiopeptide, was synthesized to treat C. difficile infections. The aqueous solubility of this analog was enhanced in comparison with 4-aminothiazolyl analogs of the antibiotic natural product GE2270 A. Pharmacokinetic studies of the infected hamster were performed, and LFF571 solution (prepared with PEG-400 or Cremophor^®EL, two solubilizing agents) was administrated at a dosage of 20 mg.kg⁻¹ (55). A very low bioavailability was observed (as expecting to treat local C. difficile infection) and no detectable levels of C. difficile toxins A and B were measured in cecal content (animals were successfully treated). A randomized clinical study (phase II exploratory study in USA and Canada) was performed to compare LFF571 and vancomycin safety and efficacy (56). Moreover, a study was conducted to evaluate the safety, tolerability, and pharmacokinetic of LFF571 in healthy human volunteers (57). In both studies, LFF571 was well-tolerated in patients and in comparison with vancomycin administration, and the frequencies of serious adverse events were similar. Unfortunately, this phase II study was discontinued in 2019.

Moreover, to date, a few small peptides are on the market: peptides with good stability and high potency. These peptides are administrated with conventional forms [composed of active pharmaceutical ingredient (AMPs) and excipient(s)]. They include solid dosage forms such as tablets, capsules, powders, granules, lozenges, and liquid dosage forms such as solutions, emulsions, suspensions, and syrups. For example, vancomycin is administered by the oral route for both local and systemic delivery (Table 1). For local delivery, vancomycin is formulated in 125- and 250-mg capsules or in powder for oral solution to treat the infection of the intestinal mucosa (pseudomembranous colitis induced by C. difficile). Indeed, after oral administration, vancomycin is not usually absorbed into the blood and is excreted almost exclusively in the feces. However, absorption is reported in patients with inflammatory disorders of the intestinal mucosa (47). Nisin containing pectin/hydroxypropyl methyl cellulose (HPMC) compression-coated tablets was formulated for the treatment of local colon diseases such as irritable bowel syndrome, inflammatory bowel disease, and ulcerative colitis (58). Authors propose to coat a tablet with pectin and HPMC (two hydrophilic polymers, which swell to form a hydrogel layer upon contact with aqueous media) to form an enzymatically controlled delivery system. Adding pectin limits the hydration and swelling of HPMC that allow to maintain tablet integrity and thus nisin stability. Furthermore, in vivo studies with pectin/HPMC-coated tablet formulation conducted in healthy volunteers have demonstrated an interesting interplay between the tablet position in the gut, the hydration of the matrix, and the subsequent release pattern. Thus, HPMC maintains tablet integrity until the colon (59). For surotomycin (CB-315), peptides were proposed to treat diarrhea with severe C. difficile infection, tablet, and age-appropriate oral solid formulation that can be dispersed or dissolved. During clinical trials phase I, an insignificant absorption of surotomycin was observed and the primary route of elimination was in the feces following oral administration (60). Throat lozenge pharmaceutical forms were also developed to administer tyrothricin by the oral route in the treatment of patients with acute pharyngitis. Finally, liquid forms prepared from powders were used. Indeed, teicoplanin, to treat diarrhea with severe C. difficile infection, is presented as powder for oral solution at the dosages of 100, 200, and 400 mg. During pharmacokinetic studies, when teicoplanin is administered by the oral route at a 250- or 500-mg single dosage to healthy subjects, it was demonstrated that teicoplanin was not detected in the serum or urine but only recovered in feces (about 45% of the administered dosage) as unchanged medicinal product (44). Colistin sulfate was administered orally as a suspension via a nasogastric tube in the gastrointestinal tract for selective digestive tract decontamination in intensive care units (43).

Despite limited oral absorption of peptides, some of them are still administered orally and indicated for systemic bacterial or viral infections. For example, capsules (200 mg) of a HIV protease inhibitor, atazanavir (ATV), are on the market. ATV has a low oral bioavailability, which can be enhanced clinically by a co-administration with ritonavir and food (Table 2). To avoid food and ritonavir co-administration uses, amorphous solid dispersion system of ATV was also prepared with sodium lauryl sulfate as a carrier and polyoxylglycerides (Gelucire^® 50/13) as an absorption enhancer (Table 2). ATV solid dispersion showed a 4.7-fold increase in bioavailability compared with ATV alone (61).

Currently, vancomycin was administered by the oral route only for local C. difficile treatment. Indeed, after oral administration, during pharmacokinetic studies in human adults and during multiple dosing of vancomycin hydrochloride capsule at 250 mg every 8 h for seven dosages, no blood concentration was detected (74). So, to extend the use of vancomycin, pharmaceutical forms have been studied to improve its oral bioavailability. For example, multiple emulsion (water-in-oil-in-water) was proposed to administer vancomycin (as the model drug). The emulsion incorporating C18 unsaturated fatty acid oil (linoleic acid or linolenic acid) has improved the bioavailability more than 40% after emulsion administration into the rat colon loop (at a dosage of 5 mg.kg^–1 of vancomycin) (69) (Table 2). On the other hand, for emulsion incorporating C18 unsaturated fatty acid oil (oleic acid) or docosahexaenoic acid, enteral bioavailability was lower (more than 25%) (69). Similarly, the formulation of a solution with a co-administration of the surfactant as an absorption promoter [PEG-8 caprylic/capric glycerides (Labrasol^®) and D-α-tocopheryl polyethylene glycol 1,000 succinate (TPGS)] was studied (70). Vancomycin solutions were administered at a dosage of 20 mg.kg^–1 into the rat ileum. Formulation containing 50% of Labrasol^® and 12.5% of TPGS increased the AUC_0−6h value of the vancomycin about 2.4 times in comparison with the formulation with 50% of Labrasol^® without TPGS (70).

Thus, the excipients that are used in pharmaceutical forms have demonstrated their importance to improve the absorption of AMPs. In addition, the stability, biocompatibility, safety, and efficacy of AMPs can be further improved through novel formulation strategies and design of new DDSs.

Self-emulsifying DDS (SEDDS) and self-nanoemulsifying DDS (SNEDDS) are defined as the isotropic mixtures of oil, surfactants, and co-solvents. They present advantages for oral drug delivery like the protection against enzymatic degradation, reduced first pass metabolism, exhibiting mucus permeating properties, and enhanced absorption (89). Accordingly, daptomycin was incorporated into SEDDS that was prepared with tricaprylin (Dermofeel^®MCT) and mono-diglyceride of medium chain fatty acids (mainly caprylic and capric) (Capmul^® MCM) as oil and PEG-40 hydrogenated castor oil (Cremophor^® 40) as the surfactant. Daptomycin was studied for the treatment of complicated skin infections, bacteremia, and right-side endocarditis caused by multiresistant Gram-positive bacteria. In vitro mucus permeation study demonstrated that SEDDS formulation improved daptomycin permeation by a factor 2 in comparison to pure daptomycin and protected daptomycin against enzymatic degradation (90). Similarly, SEDDS containing vancomycin, 25% of glycerol monocaprylate (Capmul^® 808G), 37.5% of PEG-40 (Cremophor^® R40), 13.6% of diethylene glycol monoethyl ether (Transcutol^® HP), and 26.5% of dimethyl sulfoxide (DMSO) was developed. SEDDS formulation improved intestinal in vitro mucosa permeating properties, nearly 45% of vancomycin permeated the mucus in SEDDS formulation within 4 h, whereas <5% permeated with free vancomycin (Table 2). Ex vivo permeation across porcine small intestinal mucosa confirmed these results (30 vs. 5% with SEDDS formulation and free vancomycin, respectively) (71). SNEDDS formulation of ATV was also developed. Optimized formulation was prepared with glyceryl monolinoleate (Maisin™35-1) as oil and diethylene glycol monoethyl ether (Transcutol^®P) as the surfactant. After oral administration into rats at a dosage of 7.2 mg.kg^–1 of ATV, the area under the curve (AUC) of ATV was improved 2.57-fold with SNEDDS compared to pure ATV (administered in the form of 0.3% carboxymethyl cellulose suspension) (62) (Table 2).

Microparticles (microspheres or microcapsules) are defined as particles with size larger than 1 μm. They are promising encapsulation systems for protecting peptides from degradation, enhancing peptide stability, and providing an increased surface to volume ratio for peptide release and gastrointestinal absorption (91). In this way, oral polymeric microparticulate as a carrier of polymyxin B was developed. Polymyxin B, which has a potent bactericidal activity against a broad range of Gram-negative bacteria (e.g., Pseudomonas aeruginosa), is not absorbed orally. After encapsulation into crosslinked alginate/chitosan microparticles, the biological activity of polymyxin B was conserved due to microparticle stability in a gastric environment (Table 2) (65–67). Moreover, these microparticles demonstrated their ability to target the gut-associated lymphoid tissue (GALT) by Peyer's patch uptake, but more experiments have to be performed to demonstrate an eventual improvement of polymyxin B absorption (67).

Liposomes (vesicles in which an aqueous volume is entirely surrounded by a bilayer phospholipid membrane) are the first nanoparticulate delivery systems that reached the market (92). Conventional formulations of liposomes were instable in the gastrointestinal environment, but the modification of their composition by incorporating a specific phospholipid (e.g., DSPC, DPPC, and tetraether lipids) (92, 93) or by polymer coating (e.g., chitosan and PEG) (93, 94) enhanced their stability. Recent research on oral delivery systems has shown that liposomes can be employed to improve the bioavailability of encapsulated materials by protecting them against the chemical or enzymatic degradation environment and by improving their intestinal absorption (95). Uhl et al. (73) prepared a vancomycin-loaded liposome containing glycerylcaldityltetraether lipid (TEL). For in vivo studies, different vancomycin formulations were administered by gavage in Wistar rats. The obtained bioavailability 1 h after oral administration demonstrated that a 3-fold increase of vancomycin was observed by using TEL-liposome. Anderson et al. (72) formulated another vancomycin-loaded liposomes coated with folic acid to target intestinal epithelial cells expressing folic acid receptors. After the oral administration of vancomycin formulations (61.75 mg.kg⁻¹) in Sprague–Dawley rats, the absolute bioavailability was 1.74, 6.7, and 21.8% from vancomycin solution, uncoated liposome, and folic acid-coated liposome, respectively. Thus, the folic acid and the liposomal formulation increase by a 12.5-fold the vancomycin bioavailability compared to vancomycin solution. In the same way, Arregui et al. (64) prepared a proliposome formulation [defined as dry, free-flowing particles coated with phospholipids, which can immediately form a liposomal suspension when in contact with water (96)] containing diacetyl phosphate and stearylamine to increase drug loading and to enhance the oral absorption of daptomycin. Pharmacokinetic studies in rats demonstrated that, after the oral administration of daptomycin at a dosage of 40 mg.kg^–1, a greater AUC_0−t (46.39 ± 5.69 μg/ml/h), and higher C_max (8.35 ± 0.64 μg/ml) were observed with proliposomal compared to free drug (AUC_0−t and C_max were less than the limit of quantification) (Table 2).

Nanoparticles (including nanospheres and nanocapsules) are lipid- and polymer-based nanocarriers. These nanoparticle approaches have some advantages for oral administration by protecting the encapsulated drug from enzymatic degradation, facilitating mucus diffusion, and membrane permeation (45). All these systems have already demonstrated their ability to improve peptide bioavailability (97–99).

Concerning AMPs, niosomes were developed. Niosomes (lipid-based nanoparticles) are similar to liposomes, but a bilayer is formed by a non-ionic surfactant and stabilized by the addition of cholesterol. These particles show a high stability in the gastric environment and a high permeability across the intestine (100). Polymyxin B niosomes were prepared using sorbitan monostearate (Span^® 60) and cholesterol. Vesicles were stable in simulated gastric and intestinal fluids, and about 86.2 and 78.5% of polymyxin B were retained, respectively (Table 2). Pharmacokinetic studies (at 2.0 mg.kg^–1 dosage of polymyxin B in rats) demonstrated that polymyxin B niosome administrated orally present pharmacokinetic parameters (AUC₀₋₄₈, t_1/2) similar to polymyxin B sulfate administrated intravenously (68).

Polymeric nanoparticles provide also the possibility to enhance oral absorption. Eudragit^® RL 100 [a copolymer of poly (ethylacrylate and methyl-metacrylate)] nanoparticles were prepared by nanoprecipitation to encapsulate ATV. During in vivo pharmacokinetic studies in rats (at 7.2 mg.kg⁻¹ dosage of ATV), the values of C_max and AUC₀₋₂₄ increased to 1.1- and 2.91-fold, with pure drug and optimized nanoparticle formulation, respectively. These results demonstrate the considerable performance of the nanoparticulate DDS in enhancing the bioavailability of ATV (63).